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PAXgene Tissue RNA/miRNA Kit

For isolation and purification of total RNA, including miRNA, from tissues fixed and stabilized using the PAXgene Tissue System

  • Effective purification of high and low molecular weight RNA from tissue with preserved morphology
  • Protocols for paraffin-embedded or paraffin-free tissues
  • Minimal genomic DNA contamination

Feature

Specification

Format

Spin column

Technology

Silica membrane

Main sample type

Human tissue

Sample amount

  • Up to 10 mg from PF* tissue
  • 2–5 sections 10 µm thick from PFPE** tissue with a maximum
  • size of 15 x 15 mm

Elution volume

14–40 µl

Time per run

  • 70 min + 15 min incubation/8 samples
    (120 min for fibrous tissues)

Processing

Manual: centrifugation

* PAXgene Tissue-fixed
** PAXgene Tissue-fixed, paraffin-embedded


Intended Use

The PAXgene Tissue RNA/miRNA Kit is intended for the purification of total RNA, including miRNA, from PAXgene Tissue-fixed (PF) and PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples. The kit is intended to be used as part of the PAXgene Tissue System, which additionally includes the PAXgene Tissue FIX Container and the PAXgene Tissue STABILIZER Concentrate for the collection, fixation, stabilization and storage of tissue samples.

For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.

Order information

Product

Catalog Nr.

Price

PAXgene Tissue RNA/miRNA Kit (50)

For 50 RNA preps: PAXgene RNA MinElute Spin Columns, PAXgene Shredder Spin Columns, Processing Tubes, Microcentrifuge Tubes, Carrier RNA, RNase-Free DNase, and RNase-Free Buffers. To be used in conjunction with PAXgene Tissue FIX Containers + PAXgene Tissue STABILIZER reagent.

766134
562 USD

Details

Performance

Total RNA isolated with the PAXgene Tissue RNA/miRNA Kit is highly pure. Genomic DNA contamination is minimized and the purified RNA is ready to be used in downstream applications with no detectable inhibition of PCR. The resulting eluate includes smaller RNA molecules, such as 5.8S rRNA, 5S rRNA, tRNAs and miRNAs. Studies have shown that miRNA purified from PFPE tissue samples is reliably quantified, with high concordance to flash-frozen samples (Figure 1).

Principle

The PAXgene Tissue RNA/miRNA Kit enables purification of total RNA from tissues fixed and stabilized using the PAXgene Fixation and Stabilization products, which preserve tissue morphology and biomolecule integrity by avoiding destructive crosslinking and degradation found in formalin-fixed tissues. The purified RNA and miRNA have no inhibitory chemical modifications and thus, can be used in sensitive downstream applications (Figure 2). 

Procedure

PAxgene Tissue-fixed (PF) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples are disrupted and homogenized in binding buffer. After centrifugation to remove cellular debris, optimal conditions are created for binding of RNA molecules to the silica membrane. Contaminants are then washed away and DNase I treatment removes any trace amounts of bound DNA. Total RNA, including miRNA, is then eluted in a low-salt elution buffer and denatured by heating (Figure 3).

The PAXgene Tissue RNA/miRNA Kit provides 2 protocols for purification of RNA, including miRNA, from different starting materials:

  • Sections of PFPE tissues
  • PF tissue samples (without paraffin embedding)
Applications

Total RNA and miRNA purified with the PAXgene Tissue RNA/miRNA Kit is ready to be used in a range of downstream research applications (see Resources), including:

  • cDNA synthesis
  • Gene expression arrays
  • End-point RT-PCR
  • Quantitative RT-PCR
  • Detection and quantification of miRNA
  • RNA sequencing
     
RNA, including miRNA, purified from mirrored human breast cancer tissue fresh frozen in liquid nitrogen using the QIAGEN miRNeasy Kit, or from sections of PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue using the PAXgene Tissue miRNA Kit. Shown is a scatterplot of Ct values from different single miRNA-specific RT-qPCR assays using the QIAGEN miScript System: miR9, -10a, -10b, -29a, -103, -125b, -143, -145, -192 and miScript PCR controls RNUA1, RNU5A, RNU6B, SNORD25, SCARNA17, SNORA73A; R2: coefficient of determination.Zoom image

Figure 1. High concordance of miRNA expression between total RNA isolated from PFPE and flash-frozen tissue.

RNA, including miRNA, purified from mirrored human breast cancer tissue fresh ...
SYBR Green real-time RT-qPCR was performed with 10 ng RNA from cryopreserved, formalin-fixed, paraffin-embedded (FFPE) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue (modified according to Groelz et al. (2013) Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality. Exp. Mol. Path. 94, 188). Depicted are the average delta-Ct values (delta-Ct = Ct[FFPE] – Ct[cryo] or delta-Ct = Ct[PFPE] – Ct[cryo]) from 6 different assays with amplicons ranging from 109 to 465 bp.Zoom image

Figure 2. RNA purified without chemical modification from PFPE tissue using the PAXgene Tissue RNA/miRNA Kit

SYBR Green real-time RT-qPCR was performed with 10 ng RNA from cryopreserved, ...
Disruption and homogenization of the tissue sample is performed in binding buffer, Buffer TM1. After centrifugation to remove residual cell debris, isopropanol is added to the lysate to provide appropriate binding conditions for all RNA molecules 18 nucleotides and longer. The sample is then applied to a PAXgene RNA MinElute spin column, where total RNA binds to the membrane and contaminants are efficiently washed away. Between the first and second wash steps, the membrane is treated with DNase I to remove trace amounts of bound DNA. After the wash steps, RNA, including miRNA, is eluted in a low-salt elution buffer and denatured by heating.Zoom image

Figure 3. The PAXgene Tissue RNA/miRNA Procedure.

Disruption and homogenization of the tissue sample is performed in binding buffer, ...

Resources

PAXgene Tissue RNA/miRNA Kit

Purification and quality of biomolecules from PAXgene Tissue-treated samples

1. Is it possible to use formalin-fixed, paraffin-embedded (FFPE) kits and protocols to isolate biomolecules from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?
No. Procedures developed for the extraction of biomolecules from FFPE tissues include prolonged proteinase K digestion and heating steps to remove chemical modifications introduced by formalin. Since the PAXgene Tissue System does not chemically modify biomolecules, these steps are not necessary and, in fact, lead to degradation of biomolecules. Instead, use dedicated PAXgene Tissue Kits and supplementary protocols for extraction of biomolecules from PAXgene Tissue-treated samples.


2. What is the purity of nucleic acids extracted with the PAXgene Tissue Kits?
The PAXgene Tissue DNA and RNA/miRNA Kits are based on proven QIAGEN technologies. Nucleic acids isolated with these kits are generally of high purity.

On average, measurements of the A260/A280 ratio for DNA purified with the PAXgene Tissue DNA Kit are >1.7, and ratios for RNA, including miRNA, purified with the PAXgene Tissue RNA/miRNA Kit are >1.8.

3. What is the average RNA yield, including miRNA, from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?    
RNA yield, including miRNA, depends on several parameters, such as tissue type, time from resection until fixation, fixation time, processing protocol used and age and storage conditions of the PFPE block.

In a study with PFPE tissue sections (area: 100 mm²;  thickness: 10 µm) median RNA yield from rat liver was 4.2 µg (n=58), from kidney 2.2 µg (n=58), from spleen 4.7 µg (n=58), from intestine 4.7 µg (n=58) and from lung 0.9 µg (n=58). See the Technical Note "Yield, purity, and integrity of RNA purified from PAXgene Tissue fixed, paraffin-embedded (PFPE) rat tissue" under Resources.    

4. How well is RNA integrity preserved in PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?  
Similar to yield, RNA integrity depends on several parameters, such as tissue type, time from resection until fixation, fixation time, processing protocol and age and storage conditions of the PFPE block. For examples of RNA integrity values from rat tissues under ideal workflow conditions, see Groelz et al. 2013under References. For examples of RNA integrity from clinical samples, see Viertler et al. 2012.

5. What are the yield and integrity of nucleic acids isolated from blocks of PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues?    
DNA and RNA isolated from PFCE tissue specimens are of high quantity and quality, comparable to PFPE tissue.    


6. Are special kits and protocols required for the isolation of biomolecules from PAXgene Tissue fixed, cryo-embedded (PFCE) tissues?    
No. Regular PAXgene Tissue Kits can be used for the isolation of RNA, miRNA and DNA from PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from PFCE samples are available under Resources.


7. Which kits and protocols can be used for the isolation of nucleic acids from microdissected PAXgene-Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) specimens?    
Regular PAXgene Tissue Kits can be used for the isolation of RNA, miRNA and DNA from microdissected PFPE and PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from microdissected PFPE and PFCE samples are available under Resources.    


Molecular analysis of biomolecules purified from PAXgene Tissue-treated samples    

1. What is the RT-PCR performance of RNA purified from PAXgene Tissue-fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues compared to RNA from snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissues?    
RNA, including miRNA, purified from PFPE and PFCE is free of chemical modifications and performs similarly or identically to RNA isolated from frozen tissue. For examples of the correlation of gene expression levels in snap-frozen tissue, FFPE and PFPE, see Figure 4 in Groelz et al. 2013and Figure 3 in Viertler et al. 2012 under Resources.

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